TRC8 represents the first rearrangement of any protein that interacts with VHL and its function may be germane to the development of renal cell carcinoma (RCC). The TRC8 gene was identified by positional cloning of a t(3;8) translocation in a family with hereditary RCC and non-medullary thyroid cancer. Affected individuals lacked germline VHL mutations although the morphology of the RCCs was identical. VHL functions as an F-box protein in an SCF-related (VCB) ligase complex that targets HIFalpha for ubiquitination. TRC8 is located in the ER and contains a RING-H2 finger with E3-ubiqutin ligase activity. At least part of the function of VHL is believed to involve the ER in an alternative (non-VCB) ubiquitin ligase complex. To identify a TRC8 function, we isolated the Drosophila homologue, DTrc8, and exploited genetic approaches in flies. Loss of DTrc8 and DVhl result in identical midline phenotypes, and Trc8 physically and genetically interacts with Vhl. From a 2-hybrid screen, we found that human and Drosophila TRC8 interact with the MPN domain of JAB1/CSNS, a component of the COP9 Signalosome which regulates SCF function by removing NEDD8 from Cullins. Jab1/Csn5 has deneddylase activity, and JAB 1 has also been shown to regulate nuclear-cytoplasmic transport and proteasome-mediated degradation of p27 kip1, which is characteristically abnormal in RCCs and which has been linked to VHL mutations. Overexpression of DTrc8 is growth suppressive with several distinct phenotypes, and overexpression of Trc8 and Vhl together produces a unique wing phenotype. Genetic crosses in flies demonstrate that the effects of Trc8 overexpression are corrected by cyclin E and exacerbated by the p27kipl-like molecule, Dacapo. Thus, we hypothesize that the Trc8 overexpression phenotypes are dependent upon the interactions of Trc8 with Vhl and Jab1/ Csn5. We also hypothesize that overexpression of human TRC8 will have similar consequences in mammalian cells. Our overall strategy is to define the interaction domains and introduce point mutations that disrupt them. Mutant proteins will be tested in transgenic flies for: 1) alterations in phenotypes resulting from Trc8 overexpression; 2) biochemical properties of the Signalosome (deneddylation and protein complexes) and, 3) effects on defined Jab1/Csn5 mutant phenotypes. Naturally occurring VHL mutations from RCCs will be tested for their effects on TRC8 binding, ER localization and function, and the assembly of VHL complexes. Adenoviruses expressing wt and mutant TRC8 will be analyzed in mammalian cells for effects on Cullin deneddylation, ubiquitination of a VHL-GFP fusion, cell-cycle parameters and levels of regulatory factors. In our final Aim, mitotic recombinant clones expressing wt or mutant DTrc8 will be used for an independent analysis of Trc8 function (and results integrated into the mammalian analysis). Lastly, because wt DTrc8 induces Minute-like phenotypes in adult sensory hairs, the MPN domains of eIF3h and eIF3f will be tested for interactions and further characterized if positive.